Suppression of thromboxane levels by percutaneous administration of aspirin

ABSTRACT

A method is disclosed for inducing thromboxane suppression in a mammalian subject by percutaneously administering a pharmaceutical composition containing aspirin. Articles useful for practicing the therapeutic methods of the invention are also disclosed.

This application is a divisional of U.S. application Ser. No.08/780,426, filed Jan. 8, 1997, now issued as U.S. Pat. No. 5,763,425,which is a continuation of U.S. application Ser. No. 08/339,646, filedNov. 14, 1994, now abandoned, which is a continuation of U.S.application Ser. No. 08/047,516, filed Apr. 19, 1993, now abandoned,which is a divisional of U.S. application Ser. No. 07/899,209, filedJun. 16, 1992, now U.S. Pat. No. 5,240,917, which is acontinuation-in-part of U.S. application Ser. No. 07/680,195, filed Apr.3, 1991, and now abandoned. This application also claims priority fromPCT International Application No. PCT/US92/02576, filed Apr. 2, 1992.

FIELD OF THE INVENTION

The present invention relates to the use of acetyl salicylic acid(aspirin) as an antithrombotic agent and as an agent to treat othermedical conditions benefiting from suppression of thromboxane levels.Particularly, the present invention relates to the percutaneousadministration of aspirin for inducing such effects and treating suchconditions.

BACKGROUND OF THE INVENTION

With the recognition of the role of antithrombotic agents in clinicalmedicine, investigators have pursued their efficacy, optimal dose, routeof administration and safety. Aspirin has been found to be an effectiveantithrombotic agent in patients with cerebrovascular disease andischemic heart disease. Aspirin may also have other antithromboticapplications. Although aspirin has become widely used as anantithrombotic agent, it still exhibits undesirable side effects,including gastrointestinal toxicity which is probably dose related.

To induce its suppressive effects, aspirin irreversibly acetylates theenzyme cyclo-oxygenase found in platelets and vascular wall cells [Burchet al., J. Clin. Invest. 61:314 (1978); Majerus, J. Clin. Invest.72:1521 (1983); Roth et al., J. Clin. Invest. 56:624 (1975)].Cyclo-oxygenase converts arachidonic acid to thromboxane-A₂ (TXA₂) inplatelets and to prostaglandin-I₂ (PGI₂ or prostacyclin) in vascularwalls [see for example, FitzGerald et al., J. Clin. Invest. 71:676(1983); Preston et al., N. Engl. J. Med. 304:76 (1981)]. TXA₂ inducesplatelet aggregation and vasoconstriction, while PGI₂ inhibits plateletaggregation and induces vasodilation. In other words, aspirin can haveboth an antithrombotic effect (by reducing TXA₂ production) and athrombogenic effect (by reducing PGI₂ production). As a result, strikingan appropriate balance between aspirin's effects on TXA₂ and PGI₂production has been a goal of aspirin therapy under these circumstances.

It is generally accepted that when aspirin is administered in doses ofapproximately 1,000 mg/day, it inhibits both TXA₂ and PGI₂ synthesis[Weksler et al., N. Engl. J. Med. 308:800 (1983)]. Daily administrationof very low doses of aspirin (approximately 40 mg/day) has been reportedto inhibit thromboxane-B₂ synthesis in vitro and to reduce the urinaryexcretion of 2,3-dinor-thromboxane-B₂ (both of which are metabolites ofTXA₂), without producing significant changes in the urinary excretion of6-keto-prostaglandin-F₁ a and 2,3-dinor-6-keto-prostaglandin-F₁ a (whichare both metabolites of PGI₂ production) [Patrignani et al., J. Clin.Invest. 69-1366 (1982); FitzGerald et al., supra]. While 40 mg/day hasno significant effect on prostacyclin biosynthesis, it does have somemeasurable effect [FitzGerald et al., supra]. Moreover this dose doesnot suppress 2,3-dinor-TXB₂ very well and it is not known whether itsuppresses bradykinin-stimulated prostacyclin formation. Therefore, thisdose has not been demonstrated to provide selective inhibition ofthromboxane synthesis without also inhibiting prostacyclin formation.

In contrast, others have reported that equally low doses of aspirinreduced PGI₂ synthesis by 50% in both arterial and venous tissue[Preston et al., supra], and even lower doses (20 mg/day for 1 week)have been reported to inhibit PGI₂ synthesis in both arterial and venoustissue by 50% in atherosclerotic patients [Weksler et al., supra]. Ithas been proposed that although this differential effect on theinhibition of TXA₂ and PGI₂ synthesis has been reported when urinarymetabolites are measured to assess inhibition, there is no significantevidence for this differential effect when PGI₂ synthesis is measured byassay of vascular wall biopsy tissue or when the assays for TXA₂ andPGI₂ are performed on blood samples [Weksler et al., supra]. However, itis not possible to achieve platelet selectivity with standard oralaspirin. Inhibition of basal PGI₂ biosynthesis is similar over doses of80-2,400 mg/day and bradykinin-stimulated PGI₂ formation is abolished onoral aspirin 75 mg/day.

Aspirin has also been found to be an effective treatment for othermedical conditions which benefit from lowering of TXA₂ levels. Forexample, it has been reported that daily doses of aspirin given duringthe third trimester of pregnancy can significantly reduce the incidenceof pregnancy-induced hypertension and preeclamptic toxemia in women athigh risk for these disorders as a result of reductions in TXA₂ levels[Schiff et al., N. Engl J. Med. 321:351 (1989)]. Aspirin has also beenreported to provide positive effects in women at risk forpregnancy-induced hypertension. Low doses of aspirin were reported toselectively suppress maternal thromboxane levels, but only partiallysuppressed neonatal thromboxane, allowing hemostatic competence in thefetus and newborn [Benigni et al., N. Engl. J. Med. 321:357 (1989)]. Theuse of aspirin for reducing the risk of fatal colon cancer has also beenproposed [Thun et al., N. Engl. J. Med. 325:1593 (1991)]. Reduction ofthromboxane levels has also been suggested as a means for treatingthrombosis in patients having antiphospholipid syndrome associated withlupus [Lellouche et al., Blood 78:2894 (1991)]. Low-dose aspirin hasalso been suggested as therapy for migraine headache [see for example,Buring et al., JAMA 264(13) (1990)]. The role of arachidonic acidmetabolites (e.g., TXA₂ and PGI₂) in migraine have also been invesitaged[see for example, Parantainen et al., "Prostaglandins in thePathophysiology of Migraine" in P. B. Curtis--Prior (ed.),Prostaglandins: Biology and Chemistry of Prostaglandins and RelatedEicosanoids (new York: Churchill Livingstone 1988), pp. 386-401;Puig-Parellada et al., Headache 31(3):156 (1991); Tuca et al., Headache29(8):498 (1989); Nattero et al, Headache 29(4):233 (1989)].

The use of aspirin as a thromboxane suppressant has been hampered by itstendency to cause gastric bleeding upon traditional administration ofaspirin in oral dose form. Studies have reported that aspirin produceserythema of the gastric mucosa in approximately 80% of patients withrheumatic diseases, gastric erosions in approximately 40%, and gastriculcer in 15% [Silvoso et al., Ann. Intern. Med. 91:517 (1979)]. Aspirinapplied topically to gastrointestinal tissue damages gastric mucosa andinduces occult gastrointestinal bleeding [Croft et al., Br. Med. J.1:137 (1967)]. Intravenous administration of aspirin may also producesome effects on gastric mucosa which is less pronounced with parenteralthan with oral administration [Grossman et al., 40:383 (1961)]. Oraladministration of diluted solutions of aspirin cause considerably lessbleeding than similar doses in tablet form, and aspirin solutionscontaining antacids with sufficient buffering capacity cause nomeasurable blood loss [Leonards et al., Arch. Intern. Med. 129:457(1972)]. Enteric-coated aspirin use results in less gastric and duodenalmucosal injury than regular aspirin [Graham et al., Ann. Intern. Med.104:390 (1986)].

It would, therefore, be desirable to provide an appropriate dosage formof aspirin which will provide thromboxane suppressing effects,preferably selective thromboxane suppressing effects, and will alsoavoid the adverse side effects observed with aspirin dosage formscurrently employed in aspirin therapies.

Several reports have been made of the incorporation of aspirin into avarious analgesic preparations. U.S. Pat. No. 4,948,588 discloses theuse of ether derivatives of glycerols or polyglycerols as percutaneousabsorption accelerators. Analgesics, such as morphine, codeine andaspirin, are suggested as possible active agents for use with theseaccelerators. An example discloses incorporation of aspirin into asuppository which was administered to male rabbits.

U.S. Pat. No. 4,654,209 discloses creams containing nitroglycerine andother active ingredients. Analgesics, such as aspirin, are suggested asactive ingredients. An example makes a cream containing 5-15% aspirin byweight which was applied to the skin of the abdomen, thigh or back ofsubjects, resulting in positive blood and urine tests for the activeingredient.

U.S. Pat. No. 4,476,115 discloses analgesic compositions applied to skintogether with or subsequent to the application of a non-toxicwater-soluble sulfite compound. Examples described the preparation ofmixtures of aspirin and anhydrous sodium sulfite which was applied tothe skin of a mammal and covered with a water impervious plastic sheetheld in place by adhesive tape. Bioavailability was observed within 30to 40 minutes as evidence by increased mobility of the subject andreduction of stiffness.

Although such aspirin preparations have been used for their analgesiceffects, such preparations have not to applicant's knowledge beenapplied for therapy in which thromboxane suppression is desired.

SUMMARY OF THE INVENTION

In accordance with the present invention, thromboxane suppressingeffects are provided without the gastric side effects normallyassociated with aspirin therapy. Aspirin is applied topically to apatient's skin such that it is percutaneously absorbed. The aspirin istaken into the bloodstream in quantities sufficient to inhibit TXA₂synthesis. The methods of the present invention can be used to treat anymedical condition for which suppression of thromboxane levels isbeneficial. For example, such methods can be used to produceantithrombotic effects and to treat pregnancy-induced hypertension andpreeclamptic toxemia.

The aspirin can be applied by use of a support or carrier which containsthe aspirin preparation, including without limitation suspensions,creams, solutions, patches (adhesive and non-adhesive), gels, ointments,plasters, plaques or other known forms for applying topical agents, aslong as the aspirin can be delievered in a form which will penetrate theskin (such as, for example, in a solubilized form). Articles can be madewhich incorporate the aspirin preparation, in some instances with asupport or carrier (such as in the form of an adhesive patch), which areuseful in practicing the therapeutic methods of the present invention.

Absorption enhancing agents and other pharmaceutical carriers can beincorporated into the aspirin preparation in accordance with knownmethods. Propylene glycol, with or without isopropyl or ethyl alcohol,is a preferred carrier. In addition to aspirin, other active ingredientscan be incorporated into preparations for use in the present inventionsuch as anti-arrythmics, blood pressure regulators, etc.

The aspirin content of the preparation will vary depending on the formof administration used. In one embodiment, the aspirin is applied at 750mg/day at a concentration of about 9% aspirin. To achieve the desiredsuppression effects, aspirin is preferably administered over a period ofseveral days until TXA, levels are reduced to minimal levels, preferablyless than 50% of baseline levels, more preferably less than 10% ofbaseline levels, most preferably less than 5% of baseline levels.Although TXA₂ levels will be reduced almost immediately, substantialreductions in those levels are achieved and maintained by dailyadministration over a course of several days, preferably at least 4days, most preferably at least 10 days.

Any form of aspirin may be employed in practicing the present inventionas long as the active compound can penetrate the skin. The term"aspirin" as used herein and the apended claims is intended toencompass, without limitation, all such forms. Suitable forms mayinclude acetyl salicylate and salts, esters, hydrates, etc. thereof.Particular salts which may be used include without limitation thelactate, sodium and lysine salts of aspirin Compositions and articles ofmanufacture of the present invention may also include aspirin in a firstform (such as for example, an aspririn "prodrug" or another stabilizedcompound containing acetyl salicylate) which is later converted to asecond form which can penetrate the skin.

In studying the effects of the methods of the present invention plateletcyclooxygenase activity was used as a measure of aspirinbioavailability, in addition to plasma drug levels. A preferred vehicle,propylene glycol and ethanol, is widely used as a skin permeant and waschosen to avoid ex vivo deacetylation to the inactive metabolite,salicylate. In certain preferred embodiments, aspirin applied dailyinduced a dose-dependent inhibition of platelet cyclooxygenase, asmeasured by serum TXB₂. Maximum inhibition was achieved at 10 days andexceeded 95% at the highest dose. Such a degree of suppression ispreferred to sufficiently inhibit platelet function and TXA₂biosynthesis in vivo. Inhibition of urinary TX-M followed a similarpattern. TX-M is a major enzymatic metabolite of TXB₂ and its excretionis an index of TXA₂ biosynthesis in vivo. In contrast, the vehicle alonehad no effect on serum TXB₂ or urinary TX-M. Following withdrawal oftherapy, serum TXB₂ and TX-M recovered gradually over a period of days.This is consistent with inhibition of platelet cyclooxygenase in vivo.As the enzyme is inhibited irreversibly, recovery of platelet TXA₂biosynthesis parallels the formation of new platelets, a process thathas a half-life of 5 days.

In contrast to the marked inhibition of TXA₂, there was littleinhibition of basal or stimulated PGI₂ formation. Basal PGI-M excretion,an index of in vivo PGI₂ biosynthesis, decreased 24% by day 4 on thehighest dose of dermal aspirin. No further inhibition occurred despitecontinued application and by day 10, PGI-M excretion remained at 83% ofbaseline. This may reflect the contribution of platelet endoperoxides toPGI₂ biosynthesis or local inhibition of PGI₂ biosynthesis. PGI₂formation in response to bradykinin infusion was also unaltered. Incontrast, oral aspirin 75 mg/day suppressed basal andbradykinin-stimulated PGI-M excretion, as previously demonstrated.

The preservation of vascular cyclooxygenase is consistent with the lowbioavailability of the dermal aspirin. Plasma aspirin and salicylatewere determined using a highly sensitive assay that can measure levelsof <0.1 ng/ml. Following oral aspirin 325 mg or 162 mg, peak plasmaaspirin levels were 2.0 and 1.3 ug/ml, respectively. In contrast,following dermal aspirin, plasma levels peaked at 237±114 ng/ml andplasma salicylate peaked at 788±114 ng/ml.

These data suggest that aspirin applied to the skin is absorbed veryslowly, resulting in a delayed onset and offset of activity. Plateletspassing through the site of application are inhibited by relatively highconcentrations of aspirin. A similar localized platelet effect has beenreported with oral aspirin, where inhibition of serum TXB₂ occurs priorto the appearance of aspirin systematically. As platelet cyclooxygenasecannot recover, cumulative inhibition of all platelets occurs over time.In contrast, little aspirin reaches the systemic circulation, so thatvascular cyclooxygenase is protected. The poor systemic bioavailabilityof dermal aspirin presumably reflects low skin permeability and dilutionand inactivation in the venous and pulmonary circulations.

Although in one subject there were no histological changes following 10days of drug application at 250 mg/day, skin reactions were noted in 30%of the subjects, including erythema and peeling. Similar reactions occurwith high concentrations of salicylate. Preliminary studies show thatreactions may be avoided by alternate day application. Such regimenshave been used without reactions for up to 8 weeks. Alternatively,modifications to the preparation, such as using the lactate or sodiumsalt of aspirin, or the vehicle, may be better tolerated. Lowerconcentrations and smaller doses may be feasible under occlusiveconditions, which enhance drug absorption.

When the methods of the present invention are used, as demonstratedfurther below with respect to certain preferred embodiments, aspirin isabsorbed through the skin and results in marked and selective inhibitionof platelet cyclooxygenase. This approach may prove useful in patientswith known peptic ulcer disease or during coincident administration ofanticoagulants, such as Warfarin or heparin. The methods of the presentinvention should be particularly helpful in co-administration withwarfarin as the high incidence of bleeding associated with oral aspirinand Warfarin is gastrointestinal and thought to be secondary to the oralaspirin effect.

Use of such aspirin preparations in accordance with the presentinvention provides thromboxane suppression effects, preferably selectivesuppression effects, without exposing the gut to high localconcentrations of aspirin, which should permit its use in patients with,for example, gastric intolerance, or duodenal or gastric ulcers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of serum TXB₂ (ng/ml) levels during administration ofdermal aspirin 250 mg/day and 750 mg/day vs. vehicle for 10 days andfollowing withdrawal of therapy.

FIG. 2 is a graph of urinary excretion of 2,3-dinor TXB₂ (TX-M),expressed as a percent of baseline, during the administration of dermalaspirin or vehicle for 10 days and following withdrawal of therapy. Notethat the baseline levels were 381±48, 440±56 and 498±76 pg/mg creatininefor vehicle, aspirin 250 mg and aspirin 750 mg groups, respectively.

FIG. 3 is a graph of urinary excretion of 2,3-dinor-6-keto PGF₁₂(PGI-M), expressed as a percent of baseline, during the administrationof dermal aspirin or vehicle for 10 days and following withdrawal oftherapy. Note that the baseline levels were 244±68, 402±139 and 248±73pg/mg creatinine for vehicle, aspirin 250 mg and aspirin 750 mg groups,respectively.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The advantages of the present invention can be appreciated by referenceto the following example which is meant to illustrate, but not limit,the present invention.

EXAMPLE 1

Five healthy adult volunteers (3 male, 2 female) were studied. Eachrefrained from ingesting oral aspirin for two weeks prior to study.Prior to treatment in accordance with the present invention, baselinethromboxane levels and hemoccults were obtained.

Thromboxane levels were measured by assaying for blood levels ofthromboxane-B₂ in accordance with the method described in Braden et al.,Circulation 82:178 (1990). Thromboxane levels can also be measured ineither blood or urine according to known methods which include thosewithout limitation disclosed in the following references: Hirsh et al.,supra; Robertson et al., N. Engl. J. Med. 304:998 (1981); Pedersen etal., N. Engl. J. Med. 311:1206 (1984); Patrignani et al., J. Clin.Invest. 69:1366 (1982); Preston et al., 304:76 (1981); Hirsh et al., N.Engl. J. Med. 304:685 (1981).

Salicylate levels were determined according to the following method. Theprocedure for determining salicylate is based on the formulation of aviolet colored complex between ferric iron and phenols. Substances otherthan salicylate may react to give a positive test, but false negativeresults do not occur. The color reagent contains acid and mercuric ionsto precipitate protein. References relevant to the assay method include:Trinder, Biochemical Journal 57:301 (1954); Tietz, Fundamentals ofClinical Chemistry, W. B. Saunders Co., 1970, pp. 882-884; Meites,Pediatric Clinical Chemistry, A.A.C.C., 1977, p. 192.

Trinder's Reagent was prepared as follows. 40 gm of mercuric chloridewas dissolved in about 700 ml of deionized water by heating. Thesolution was cooled and 120 ml of 1N HCl and 40 gm of ferric nitrate,Fe(NO₃)₃ 9H₂ O, were added. When all the ferric nitrate had dissolved,the solution was diluted to a total volume of 1000 ml with deionizedwater. This stock solution is stable for approximately one year.

Standards were prepared as follows. A Stock Standard (200 mg/100 ml) wasprepared by dissolving 464 mg of sodium salicylate in deionized waterand diluted to a total volume of 200 ml. A few drops of chloroform wereadded as a preservative. This standard solution is stable forapproximately 6 months under refrigeration. 5, 10, 25, and 40 ml ofstock standard were pipetted into a series of 100 ml volumetric flasks,diluted to a total of 100 ml with deionized water, and mixed.

0.2 ml of serum or heparinized plasma were used as sample specimens. 0.2ml of each standard and each sample was pipetted into respectivelylabeled disposable polystyrene tubes. Into another polystyrene tube, 0.2ml of deionized water was pipetted to be used as a reagent blank. 1.0 mlof deionized water was added to all tubes. 1.0 ml of Trinder's reagentwas then added to all tubes, which were mixed and let stand tubes for 5minutes. The tubes were then centrifuged for 10 minutes. The clearsupernatant (minimum of 1.0 ml) was placed into respectively labeled10×75 nm cuvettes. Samples were analyzed by reading % T at 540 nmagainst the reagent blank set at 100% T. Sample values were comparedwith standard values to determine levels. Results over 75 mg percentwere diluted and re-analyzed.

A preparation of aspirin in isopropyl alcohol and propylene glycol wasprepared by mixing "Aspirsol"™ topical aspirin (NDC54102-001-01;commercially available from TERRI Pharmaceuticals, Inc., PO Box 6454,Kingwood, Tex. 77325) in accordance with the package instructions exceptthat 8 ml instead of 10 ml of the suspending solution was used. Theresulting solution contained approximately 9% aspirin rather than the7-8% indicated on the package label.

After base levels of thromboxane had been measured, the aspirin solutionwas first applied on the morning of Day 1 to the skin of the humansubjects within an hour of mixing by rubbing the aspirin solution on thearms and/or chest of the subject. The application was repeated withfreshly prepared solution for each of four additional mornings (Days2-5) in the same manner. Between applications the subjects followedtheir normal schedule of bathing and showering. Eight hours after thefifth application (on Day 5) blood was drawn for salicylate levels andthromboxane levels to be determined. With one subject, blood was drawnfor TXA₂ levels every day just before application of a new aspirinsolution (i.e., approximately 24 hours after application of the previousaspirin solution). Hemoccults were also tested.

Two of the subjects continued daily application for another five days(total ten days). For Days 6 and 7, aspirin solutions freshly preparedas described above were used. Beginning with Day 8, a different aspirinpreparation was used. This second preparation was prepared by crushingaspirin tablets containing approximately 975 mg aspirin to form apowder. The powder was then formed into a paste with approximately 2 mlof distilled water. This paste was then mixed with 4 ml propylene glycoland 4 ml ethanol to produce approximately 10 ml of a cloudy solution.This cloudy solution was then filtered to remove excipients and otherinsoluble material found in the crushed aspirin tablets. Afterfiltering, approximately 10 ml of a clear solution was obtained whichcontained approximately 9% aspirin. 8 ml of the resulting solution wasused in each application. This second solution was applied to the twocontinuing subjects as previously described. Salicylate and thromboxanelevels were checked after the tenth day.

Thromboxane levels are summarized in Table 1.

                  TABLE 1                                                         ______________________________________                                        Thromboxane Levels                                                              (ng/cc)                                                                       Subject  Baseline Day 2 Day 3 Day 4  Day 5 Day 10                           ______________________________________                                        1      401      311     250   199    105/35.sup.1                                                                        18                                   2 372    105 12                                                               3 .sup. 107.sup.2    124                                                      4 402    152                                                                  5 394     25                                                                ______________________________________                                         .sup.1 The first number is the level measured in the morning of Day 5         prior to administration of the new Day 5 dose. The second number is the       level measured eight hours after the Day 5 application.                       .sup.2 Subject 3 had a very low measured baseline thromboxane level which     is believed to have been a sampling error.                               

As summarized in the table, baseline thromboxane levels were found torange from 372-402 ng/cc in four out of five subjects. The low baselinefor Subject 3 is believed to be erroneous and, as a result, the data forSubject 3 was not considered relevant. After five daily applications ofaspirin in accordance with the present invention, caused a decrease inthromboxane levels of at least 50%. The two subjects that continuedtherapy in accordance with the present invention for another five dayshad marked suppression by Day 10 of 95 and 97% to levels of 18 and 12ng/cc. Salicylate levels in four of five patients on day five weremeasured as 1 mg percent or less (approx. 1 mg percent being the lowerlimit of sensitivity of the assay). All hemoccults taken were negative.No gastrointestinal symptoms or other side effects were noted orreported by the subjects.

EXAMPLE 2

Only healthy male and female volunteers were studied. The subjects wereasked to avoid aspirin and any other cyclooxygenase inhibitors for the10 days before and throughout the period of investigation. Aspirin(acetyl salicylic acid, USP) powder was dissolved in propylene glycoland either isopropyl alcohol or ethanol (1.7:1 v/v) to a finalconcentration of 94 mg/ml. Preliminary studies demonstrated that aspirinwas stable in this vehicle, with less than 1% salicylate detected after24 hr at room temperature. The aspirin preparation was made dailyimmediately prior to its application Volunteers attended the clinicwhere the preparation was applied and were asked not to wash the areafor at least 12 hours. The aspirin solution was applied to the forearmand upper arm over a 15 min interval. Volunteers received aspirin 250 mg(n=4), aspirin 750 mg (n=6) or vehicle (n=6) for 10 days and werefollowed for 8 days following drug withdrawal. The volunteers were aged31-56 years, with equal numbers of male and females in each treatmentgroup.

Blood without anticoagulant was obtained for serum TXB₂, the stablemetabolite of TXA₂, prior to and at intervals during and followingaspirin administration. The blood was allowed to clot in glass at 37° C.for 60 min and the serum removed and stored at -20° C. until analyzed.Urine was collected over 24 hours at corresponding times for measurementof 2,3-dinor-TXB₂ (TX-M) and 2,3-dinor-6-keto-PGF_(1a) (PGI-M), majorenzymatic metabolites of TXA₂ and PGI₂, respectively [Lawson et al.,Analyt. Biochem. 150:463 (1985); FitzGerald et al., N. Engl. J. Med.310: 1065 (1984)]. Excretion of these products is an index of the invivo formation of their parent compounds [FitzGerald et al., supra;Reilly and Fitzgerald, Blood 69: 180 (1987)]. Serum TXB₂ and urinarymetabolites were determined by negative ion-chemical ionization, gaschromatography-mass spectrometry (NICI-GCMS) using authentic deuteratedstandards, as previously described [Braden et al., supra].

Serum TXB₂, an index of the capacity of platelets to generate TXA₂, waswithin the normal range in all subjects prior to study, demonstratingthat none had been exposed to a cyclooxygenase inhibitor. Application ofthe vehicle alone had no effect on serum TXB₂ in 6 subjects (FIG. 1).With aspirin 750 mg/day (n=6), there was a progressive reduction inserum TXB₂ in all but one of the volunteers. In the remaining subjects,serum TXB₂ was 5±3% of baseline by day 10 of application (n=5, p=0.003;FIG. 1). Aspirin 250 mg/day induced a smaller fall in serum TXB₂, whichwas 55±11% by day 10 (n=4; p<0.01). Following the withdrawal of aspirin,serum TXB₂ increased gradually and by day 8 was 93±7 and 65±9% ofbaseline for aspirin 250 mg and 750 mg, respectively.

TXA₂ biosynthesis demonstrated a similar response. Thus, there was adose dependent reduction in the urinary excretion of TX-M. At 750 mg/dayof dermal aspirin. TX-M declined gradually and was 32±7% of baseline byday 10 (n=5; p=0.002) of drug application. By 8 days following drugwithdrawal, excretion of the metabolite had recovered to 65±9% of thepretreatment value (FIG. 2). Despite the evidence of marked inhibitionof platelet cyclooxygenase, there was only a small fall in PGI₂biosynthesis, based on urinary PGI-M determinations (FIG. 3). Althoughthe changes did not achieve statistical significance (p=0.074 by ANOVA),there was an apparent dose response relationship. Thus, urinaryexcretion of PGI-M fell to 84±4% and 76±7% of baseline on aspirin 250mg/day and 750 mg/day, respectively (FIG. 3). The peak decrease in PGI-Mexcretion occurred by day 4 on both doses, in contrast to TX-M excretion

EXAMPLE 3

In an additional 4 subjects, we examined the increase in PGI₂ formationin response to intravenous bradykinin prior to and following oralaspirin 75 mg or dermal aspirin 750 mg daily for 14 days. The protocolfor bradykinin has been described previously [Clark, N.Engl.J.Med325:1137 (1991)]. Volunteers were admitted after an overnight fast tothe Clinical Research Center. Blood samples were obtained for serum TXB₂and the subject asked to void. Through a peripheral vein, 1 liter ofnormal saline was infused over 1 hour. After a further hour, bradykininwas infused in incremental doses of 100-800 ng/kg/min, each over 15 mm.The infusion was continued at the maximum tolerated dose for a totalperiod of 2 hr. Blood pressure and heart rate were monitoredcontinuously. Urine was collected in separate 2 hr aliquots prior to,during and following the bradykinin infusion.

Previous studies have demonstrated that bradykinin increases PGI₂biosynthesis by on average 2-6 fold. In the 4 subjects studied,bradykinin induced a 5.1±6 fold increase in PGI-M excretion. Twosubjects were treated with oral aspirin 75 mg/day for 14 days and twowith dermal aspirin 750 mg/day. Both preparations caused a marked fallin urinary TX-M (TABLE 2). Oral aspirin resulted in a decrease inurinary PGI-M at rest and following stimulation with bradykinin. Incontrast, resting and stimulated PGI-M excretion was largely unalteredby dermal aspirin.

                  TABLE 2.sup.1                                                   ______________________________________                                                     Dermal Aspirin                                                                            Oral Aspirin                                           (750 mg/day) (75 mg/day)                                                                 PT 1  PT 2      PT 1   PT 2                                      ______________________________________                                        TX-M     pre ASA   220     121     136  111                                      post ASA  46  29  26  43                                                     PGI-M pre ASA 163 105 104 200                                                 (rest)                                                                         post ASA 138 149  63  96                                                     PGI-M pre ASA 1520  559 433 340                                               (stim)                                                                         post ASA 1553  601 173 132                                                 ______________________________________                                         .sup.1 The excretion of TXM and PGIM before and following dermal aspirin      750 mg/day or oral aspirin 75 mg/day for 10 days. Urine samples were          collected over 2 hr before (rest) and following the administration of         bradykinin (stim). Dermal aspirin suppressed TXM, but had not effect on       PGIM.                                                                    

EXAMPLE 4

In 4 subjects (2 male, 2 female) demonstrating a marked (>90%) decreasein serum TXB₂, plasma aspirin and salicylate were determined at timedintervals (0, 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, 12 and 24 hr)following the application of aspirin on days 1 and 14. Aspirin wasapplied in a dose of 750 mg on one limb over a 15 min interval. Sampleswere drawn from the opposite arm. Blood was withdrawn into heparin (10U/ml final concentration) and potassium fluoride (5% finalconcentration), the latter to prevent ex vivo metabolism of aspirin byplasma esterases. The plasma was separated immediately and stored at-700° C. until analyzed. Aspirin and its metabolite, salicylic acid,were measured by NICI-GCMS using deuterium-labelled analogues asinternal standards, as previously described [Clark, supra].

Plasma aspirin and salicylate levels were determined following thesingle application of dermal aspirin in five subjects who demonstrated amarked (>90%) reduction in serum TXB₂. Plasma aspirin was barelydetectable up to three hours following application when it rose to237±114 ng/ml. At six hours, it fell to 52±14 ng/ml and by 24 hours itdecreased to 4±3 ng/ml. Plasma salicylate demonstrated a similarpattern. At two hours, it was 69±20 ng/ml rising to 250±77 ng/ml atthree hours. At six hours, plasma salicylate was 774±296 ng/ml. Bytwenty-four hours, the levels had fallen to 329±84 ng/ml. The later peakin salicylate levels is consistent with its being derived from aspirin.Moreover, this prolonged elevation of plasma salicylate is to beexpected, given its longer plasma half-life. Note that levels followingoral aspirin (75 mg) are 1-2 ug/ml.

Each of the references cited in this specification is incorporatedherein by reference as if fully set forth.

What is claimed is:
 1. An article useful for suppressing thromboxanelevels in a mammalian subject by contacting the skin of said subjectwith said article comprising a preparation comprising aspirin and asupport or carrier for maintaining said aspirin in a suitable form fortopical percutaneous absorption by said skin, wherein said aspirin ispresent in an amount sufficient to reduce thromboxane levels in saidsubject by more than 50% without substantially affecting prostacyclinlevels or resulting in gastrointestinal toxicity upon application ofsaid article.
 2. The article of claim 1 wherein said aspirin is providedin the form of a salt.
 3. The article of claim 2 wherein said salt isselected from the group consisting of the lactate salt of aspirin, thesodium salt of aspirin and the lysine salt of aspirin.
 4. The article ofclaim 1, wherein said preparation comprises from about 250 mg to about750 mg of aspirin.
 5. The article of claim 1, wherein said preparationcomprises about 250 mg of aspirin.
 6. The article of claim 1, whereinsaid preparation comprises about 750 mg of aspirin.
 7. The article ofclaim 1, wherein said preparation comprises about 9% aspirin.
 8. Thearticle of claim 1, wherein said aspirin is present in an amountsufficient to reduce thromboxane levels to from less than about 5% ofbaseline levels to less than about 50% of baseline levels.
 9. Thearticle of claim 1, wherein said article is in continuous contact withsaid skin for at least 4 successive days.
 10. The article of claim 1,wherein said article is in continuous contact with said skin for atleast 10 successive days.
 11. The article of claim 1, wherein saidcarrier is selected from the group consisting of suspensions, creams,solutions, patches, gels, ointments, plasters and plaques.
 12. Thearticle of claim 1, wherein said carrier is selected from the groupconsisting of propylene glycol, isopropyl alcohol, and ethyl alcohol.13. The article of claim 1, wherein said preparation further comprisesat least one other active ingredient.
 14. The article of claim 1,wherein said preparation further comprises an agent for promotingabsorption of said aspirin.
 15. The article of claim 1, wherein saidpreparation further comprises an anticoagulant.